The ELISA procedure is a quantitative in vitro test for an antibody or antigen in which the test material is adsorbed on a surface and exposed to a complex of an enzyme linked to an antibody specific for the substance being tested for with a positive result indicated by a treatment yielding a color in proportion to the amount of antigen or antibody in the test material. The ELISA procedure is described more specifically, for one, in a book entitled Methods in Molecular Biology Vol 42, John R. Crowther, Humana Press, 1995. The antibody specific for the substance being tested for in the foregoing definition constitutes a recognition molecule. ELISA-like approaches using other recognition molecules can also be used, such as aptamers, DNA, RNA & molecular imprint polymers.
The basic definition of ELISA was expanded beyond the colometric approach, wherein color is used as an indicia, to include yielding a rate of change of voltage or current conductivity in proportion to the amount of antigen or antibody in the test material, a voltametric or amperiometric approach to detection. Patent Cooperation Treaty application PCT/US98/16714, filed Aug. 12, 1998 (International Publication No. WO99/07870), entitled “Electrochemical Reporter System for Detecting Analytical Immunoassay and Molecular Biology Procedures” (hereafter the “'16714 PCT application), claiming priority of U.S. patent applications Ser. Nos. 09/105,538 and 09/105,539”), describes both a colormetric and an electrochemical reporter system for detecting and quantifying enzymes and other bioagents in analytical and clinical applications.
The ELISA procedure has also been automated. In a prior application by the present applicants, Ser. No. 09/837,946, filed Apr. 19, 2001, entitled “Automated Computer Controlled Reporter Device for Conducting Immunoassay and Molecular Biology Procedures” (the “'946 application”), the content of which is incorporated herein by reference in its entirety, an automated analytic instrument, hereafter referred to sometimes as a biosensor instrument, is disclosed that tests whether a sample (e.g. the analyte) is or contains a respective bioagent, protein and/or nucleic acid using the ELISA technique.
The biosensor instrument described in the '946 application is computer-controlled and user-friendly, which permits relatively unskilled personnel to carry out important tests for the presence of a bioagent, protein and/or nucleic acid in a sample of suspect material. An electronic controller in the biosensor instrument, such as a programmed microcontroller, controls a series of pumps to automatically sequence pumping of the individual fluids required in the ELISA procedure into a cell (or cells) necessary to produce an electro-chemical reporter, analyzes the electro-chemical data and, ultimately, displays the concentration of the bioagent determined from that analysis. Once started, the apparatus, governed by the program of the microcontroller, conducts the test automatically without the necessity for human intervention. The biosensor instrument may be housed in a single package for easy portability; and may be either battery or line powered. The content of the prior application is referred to and incorporated herein in its entirety.
In preparation for a test of suspect material with the biosensor instrument, a solution is prepared by placing the suspect material in a water-based buffer, such as a phosphate buffered saline solution. The suspect material may have been preliminarily treated, such as by exposing the material to ultrasonic energy to break the material into multiple small clumps or even granules to ensure maximum surface area exposure of the sample in solution, or the suspect material may be used “as is” as collected.
In one of the initial steps of operation of the ELISA procedure carried out by the biosensor instrument, the sample solution, referred to as the analyte, is deposited into a container or vessel that contains a quantity of micron size magnetic beads in a saline solution (or vice-versa), referred to herein as a primary antibody solution (or “1° Ab”). The surface of those beads contains a coating of an antibody to the suspect bioagent, protein and/or nucleic acid. The particular antibodies used to coat the magnetic beads are known to bind to the bioagent, protein and/or nucleic acid of interest or of concern. That is, the antibody coating exhibits a chemical “stickiness” that is selective to specific bioagents, proteins and/or nucleic acids. The analyte and the magnetic beads are then mixed together in the liquid. Assuming that the analyte is the suspected bioagent, as example, the analyte should bind to the antibody coated on the beads, forming a 1° Ab/analyte complex.
To ensure that the analyte molecules are afforded the greatest opportunity to effectively bind with the molecules of antibody coating on a magnetic bead, it was found desirable to stir the magnetic bead solution, a slurry, before undertaking the foregoing step. Awaiting use, the coated magnetic beads are stored in the saline solution in a vessel and forms a weak slurry. Over time in storage, the force of gravity causes the beads to settle to the bottom of the storage vessel. For each analysis the contents of the bead reservoirs were, preferably, mixed to ensure that when the biosensor instrument withdrew a set volume from the storage vessel, the quantity of beads that is drawn into the reaction is also set. To stir (or mix) the coated magnetic beads the biosensor instrument pumped at least a portion of the contents from the vessel and alternately then pumped that portion back therein, doing so a number of times. That action ensured that the magnetic beads were adequately dispersed in the liquid.
Pumping of the magnetic beads increases wear and tear on the pumps and/or mixers. Further, if mixing is excessive, the beads could be damaged. That in turn limits the maximum number of doses that could be obtained before it was necessary to replenish (or replace) the beads. To maximize the time between required replenishment of the beads the duration of the each mixing cycle is minimized, trading off uniformity of bead dosage for endurance.
Electric pumps consume electrical energy during operation. They also tend to generate noise. Usually, the harder the pump must work, the greater is the noise produced and the electrical power consumed. Minimizing the number of pumps in the instrument, even reducing the number of pumps by one, enhances the ability of the instrument to operate on dry cell batteries or maximize the period over which rechargeable batteries can be used before requiring a recharge. Minimizing the number of pumps also reduces the noise generated by the instrument.
Accordingly, an object of the invention is to minimize or avoid the necessity of mixing or stirring of a saline solution of magnetic beads in order to achieve a relatively uniform dispersal of magnetic beads in a primary antibody bead system for an enzyme-linked immunoassay procedure.
Accordingly, a principal object of the present invention is to increase the uniformity of bead dosage without requiring increased mixing.
A further object of the invention is to reduce the amount of maintenance necessary to maintain pump operation in the biosensor instrument and enhance the operational life of that instrument.
And, a still further object of the invention is to minimize or eliminate the bead mixing operation in the biosensor instrument.